This application claims benefit of foreign priority from Canada patent 2,267,481, filed Mar. 30, 1999.
The present invention relates to the use of antibodies for the detection of cancer. More specifically, this invention relates to antibodies specific for NDP-kinase/Nm23 and estrogen stimulated Leucine Aminopeptidase (es-LAPase) and their use for the diagnosis of ductal carcinomas in situ (DCIS) and metastisis associated with breast cancer. The present invention also relates to a diagnostic system using an antibody to NDP-kinase together with an antibody specific for an estrogen simulated for es-LAPase to detect blood serum, plasma or tissue levels of the NDP-kinase and LAPase.
The identification and characterization of risk factors and their molecular implications in the pathophysiology of human diseases such as breast cancer is essential for designing efficient diagnostic assays and therapeutic compounds. In particular, the accurate diagnostic of cancer type and aggressiveness (staging) is critical for the selection of the optimal therapeutic strategy. Staging relies on a combination of analysis including, but not limited to, histology of the tumour, detection of blood components and detection of cellular markers.
Diagnostic assays are available for breast cancer. For example, imaging techniques such as ultrasounds and x-rays (mammographies) are widely used to detect tumours. These imaging techniques, however, suffer from a limitation in the resolution of the image which prevents the detection of tumours below a certain size. Furthermore, fibroadenomas or cysts often share similar mammographic patterns as those found in malignant invasive tumours, making it difficult to determine tumour malignancy on the sole basis of mammography. Recent studies performed by Elmore (Lancet, 1999) and Goetze (Lancet, 2000) have clearly underlined the limitations of mammographic screening.
Histological analysis of biopsies is also a common procedure for the diagnosis of breast cancer and this technique relies on identification of visible phenotypes of the cells. However, this analysis is somewhat subjective and depends on the skill of the examiner.
Flow cytometry analysis of DNA from cells obtained from biopsies can also be analysed for DNA content as a measure of diploidy of cells, which may be correlated with the presence of cancer. However, such an analysis requires biopsies and extensive processing of the sample and access to costly instrumentation.
Amongst the various risk factors associated to the onset of early events leading to breast cancer, estrogen and estrogen-like compounds with estrogenic mimicking activity remain the most important determinants in the early events and progression of breast carcinogenesis. Under normal physiological conditions, there are several tissues whereby estrogenic steroids have been shown to play a critical function. These include the development of the reproductive tract, particularly secondary organs, such as the mammary glands. In addition, estrogens are also involved in the fine regulation of bone growth, liver and cardiovascular function and the estrus cycle, most likely through the induction of cell proliferation in target tissues [Sutherland, R. L., Watts, C. K. W., and Clarke, C. L. (1998). Hormones and Their Actions: Part 1 (van der Molen, H. J., King, R. J. B., Cooke, B. A., eds) pp. 197-215, Elsevier Science Publishing B. V., Amsterdam; Shekhar, P. V. M., Werdell, J., and Basrur, V. S. (1997). Environmental Estrogen Stimulation of Growth and Estrogen Receptor Function in Preneoplastic and Cancerous Human Breast Cell Lines. J. Natl. Cancer. Inst., 89: 1774-1782]. The response of such a variety of tissues to estrogen stimulation can explain in part its active role in the development and progression of different human carcinomas and in particular of breast cancer. Without wishing to be bound by any theory, it is thought that estrogens regulate various physiological functions by being involved in both xe2x80x9cimmediate-earlyxe2x80x9d and xe2x80x9cearlyxe2x80x9d events of cell function. In this regard, it appears that immediate early events induced by estrogen lead to an increased cellular proliferation most likely through the reduction in the cell cycle by accelerating the rate at which cells progress from the G1 phase towards the S phase. Recently, it has been proposed that estrogen promotes cellular proliferation by co-activating at similar estrogen concentrations, the expression of cyclin D1-Cdk4 and cyclin E-Cdk2, two critical and potentially interrelated G1 regulatory peptides [Prall, O. W. J., Sarcevic, B., Musgrove, E. A., Watts, C. K. W. and Sutherland, R. L. (1997). Estrogen-induced Activation of Cdk4 and Cdk2 during G1-S Phase Progression Is Accompanied by Increased Cyclin D1 Expression and Decreased Cyclin-dependent Kinase Inhibitor Association with Cyclin E-Cdk2. J. Biol. Chem., 272: 10882-10894].
Estrogen-based molecular diagnostics assays are available. The most widely used such assay measures estrogen and progesterone receptors in cells obtained from biopsies. It is particularly useful to determine whether a particular type of cancer will be responsive to hormonal therapy. However, this assay is of limited utility to evaluate the stage or progression of the disease and in particular it does not permit the detection of the presence of metastasis. This assay also suffers from the fact that it is necessary to obtain a biopsy. Apart from the fact that this constitutes an invasive procedure, it requires that the tumour be large enough to be detected by palpation or with imaging devices. Such large tumours may already be at a fairly advanced stage.
NDP-Kinase activity has been related to various physiological processes including DNA and RNA synthesis, production of cyclic AMP, superoxide metabolism and activation of the enzyme complex involved in DNA repair. In general the activity of NDP-Kinase has been parallelled to cellular proliferation, i.e. enhanced cytosolic NDP-Kinase activity is detected during cell proliferation. Moreover, the nm23 gene for which RNA levels are reduced in tumorous cells of high metastic potential, possesses a high degree of homology with the gene encoding the NDP-Kinase.
The nucleoside diphosphate-kinase facilitates the intracellular conversion of both deoxy and ribonucleotide diphosphates to their phosphorylated forms. Although the majority of the nucleoside diphosphate kinase (NDP-kinase) activity is found in the cytosol of various cell types, the enzyme is also present in other cell constituents such as the multimeric microtubule protein rings, isolated plasma membranes of human and rabbit platelets, and beef brain particulate material. The wide distribution of NDP-kinase reflects its active role in various fundamental cellular processes related to nucleotide and superoxide metabolism, synthesis of cyclic AMP and in general to cell proliferation and differentiation.
Its role in cell proliferation and differention makes NDP-kinase a candidate, as a cell marker, for cancer detection. This avenue has been explored by immuno-histochemical analysis of tissue sections from tumour biopsies. However, there was no clear correlation in these studies between the presence of NDP-kinase and the presence of cancerous cells [Sawan A. et al., NDP-K/nm23 Expression in Human Breast Cancer in Relation to Relapse, Survival, and Other Prognostic Factors: an Immunohistochemical Study, Journal of Pathology January 1994; 172(1):27-34; Kobayashi S. et al., Estrogen Receptor, c-erbB-2 and nm23/NDP Kinase Expression in the Intraductal and Invasive Components of Human Breast Cancers, Japanese Journal of Cancer Research August 1992; 83(8):859-65; Srivatsa P. J. et al., Elevated nm23 Protein Expression is Correlated with Diminished Progression-Free Survival in Patients with Epithelial Ovarian Carcinoma, Gynecology Oncology March 1996; 60(3):363-72]
Other cellular markers have been studied for their correlation with cancer in general and breast cancer in particular. Among these markers serum LAPase has been found to correlate with invasiveness of breast cancer (Gupta S. K. et al., Serum Leucine Aminopeptidase Estimation: A Sensitive Prognostic Indicator of Invasiveness in Breast Carcinoma, Indian Journal of Pathology in Microbiology October 1989; 32(4):301-5]. However, variations in the level of serum LAPase is also associated with other types of cancer and other disease conditions such as systemic lupus erythematosus [Inokuma S. et al., Serum Leucine Aminopeptidase as an Activity Indicator in Systemic Lupus Erythematosus: A study of 46 Consecutive Cases, Rheumatology (Oxford) August 1999; 38(8):705-8], myocardial infarction [Gupta S. K. et al., Prognostic Significance of Serum Leucine Aminopeotidase in Myocardial Infarction with Left Ventricular Failure, Journal of Assoc. Physicians India November 1987; 35(11):760-2] and viral infections [Pancheva-haschen R. et al., Serum Leucine Aminopeptidase for Monitoring Viral Infections with Plasmacytoid Reaction, Enzyme 1986; 36(3):179-86] among others, making a diagnostic based on enzymatic assay of serum LAPase non specific. Without wishing to be bound by any theory, this non-specificity may be related to the presence of several isoenzymes of LAPase.
Clearly, there is a need for more rapid, more sensitive and less invasive methods for diagnosing and staging breast cancer. In particular, cellular markers, which reflect the presence of metastasis and markers indicative of estrogen activity are highly desirable.
The present invention provides for such a method. The method relies on monoclonal antibodies specific for NDP-Kinase and estrogen-stimulated LAPase capable of detecting these cellular markers in blood and tissues. Levels and enzymatic activity of these markers correlate with the aggressiveness in general and the presence of metastasis in patients with cancer and breast cancer in particular. This immuno-based assay may be done on blood samples or tissues from biopsies. A further advantage of the assay of the present invention is that by using two markers, the probability of false positives is greatly reduced. False positive could arise when using only one marker if blood levels of this marker are altered by other physiological conditions in the patient.
The present invention relates to the use of antibodies for the detection of cancer. More specifically, this invention relates to antibodies specific for NDP-kinase/Nm23 and estrogen stimulated Leucine Aminopeptidase (es-LAPase) and their use for the diagnosis of ductal carcinomas in situ (DCIS) and metastisis associated with breast cancer. The present invention also relates to a diagnostic system using an antibody to NDP-kinase together with an antibody specific for an estrogen simulated for es-LAPase to detect blood serum, plasma or tissue levels of the NDP-kinase and LAPase.
In one embodiment of the present invention there is provided a method of detecting breast cancer in a patient by determining the level of NDP-kinase and es-LAPase in a sample.
Also according to the present invention there is provided a method of detecting a metastatic cancer in a patient by determining the level of NDP-kinase and es-LAPase in a sample.
The present invention also relates to a diagnostic system using an antibody to NDP-kinase together with an antibody specific for an estrogen simulated LAPase to detect blood serum, plasma or tissue levels of the NDP-kinase and es-LAPase.